A cell group grown from a single cell is thought to be composed of cells each having entirely identical traits, and single cell cloning to obtain such a homogeneous cell group has become an important technology both academically and commercially. For example, in order to manufacture pharmaceuticals for which preparation of homogeneous products is a prerequisite, especially for the manufacture of a protein, it is necessary to select, isolate, and grow a single cell that produces said protein and thereby to construct homogeneous cells groups that produce said protein. Single cell cloning has, therefore, become an indispensable technology for the production of proteins, in particular recombinant proteins.
The conventional method of preparing a monoclonal antibody that requires single cell cloning has employed the so-called limiting dilution method in which spleen cells obtained from an animal immunized with an antigen and immortalized myeloma cells were subjected to cell fusion to prepare hybridomas, and each cell of the heterogeneous cell group thus prepared was cultured together with a feeder cell. As the feeder cells, at this time, spleen cells that were subjected to mitomycin or radiation treatment, in order to deprive the cells of the ability to grow, have been used.
In cases where culturing was difficult, a growth factor such as IL-6 was further added to the culture system. However, these methods had the disadvantages that the feeder cells used were not a homogeneous cell group, variation due to experimental procedures was large, pre-treatment to incapacitate the growth ability was required, and the like. Furthermore, in some cells, growth from single cells was difficult even if the feeder cell was added or a growth factor and fetal bovine serum were added.